Newly Woody Artificial Diet Reveals Antibacterial Activity of Hemolymph in Larvae of Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae).

The rearing of saproxylic insects in laboratory conditions is an important task for studying the biology of insects. Through understanding nutritional needs, it is possible to optimize beetle rearing in laboratory conditions. In this study, an artificial fungi-based diet (FD) was developed for the cultivation of the darkling beetle Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae) in laboratory conditions as a model object for studying the biology of saproxylophagous beetles. To assess the influence of the diet, a number of physiological parameters were measured, including development time, body size, and weight of all stages of the beetle's life cycle, as well as its immune status. The immune status of Z. atratus was assessed on the basis of larval hemolymph antibacterial activity against six different bacterial strains assessed using disk-diffusion and photometric tests. Our findings show that the FD reduces development time and boosts the immune status as compared to beetles reared on a standard diet (SD). Samples from FD-reared larvae had pronounced antibacterial activity as compared to samples from SD-reared larvae. This work is of fundamental importance for understanding the correlations between nutrition and development of saproxylic Coleoptera and is the first report on immune status regulation in this group of insects.

Invertebrate C1q Domain-Containing Proteins: Molecular Structure, Functional Properties and Biomedical Potential.

C1q domain-containing proteins (C1qDC proteins) unexpectedly turned out to be widespread molecules among a variety of invertebrates, despite their lack of an integral complement system. Despite the wide distribution in the genomes of various invertebrates, data on the structure and properties of the isolated and characterized C1qDC proteins, which belong to the C1q/TNF superfamily, are sporadic, although they hold great practical potential for the creation of new biotechnologies. This review not only summarizes the current data on the properties of already-isolated or bioengineered C1qDC proteins but also projects further strategies for their study and biomedical application. It has been shown that further broad study of the carbohydrate specificity of the proteins can provide great opportunities, since for many of them only interactions with pathogen-associated molecular patterns (PAMPs) was evaluated and their antimicrobial, antiviral, and fungicidal activities were studied. However, data on the properties of C1qDC proteins, which researchers originally discovered as lectins and therefore studied their fine carbohydrate specificity and antitumor activity, intriguingly show the great potential of this family of proteins for the creation of targeted drug delivery systems, vaccines, and clinical assays for the differential diagnosis of cancer. The ability of invertebrate C1qDC proteins to recognize patterns of aberrant glycosylation of human cell surfaces and interact with mammalian immunoglobulins indicates the great biomedical potential of these molecules.

Kidney-Related Function of Mitochondrial Protein Mitoregulin.

A small protein, Mitoregulin (Mtln), localizes in mitochondria and contributes to oxidative phosphorylation and fatty acid metabolism. Mtln knockout mice develop obesity on a high-fat diet, demonstrating elevated cardiolipin damage and suboptimal creatine kinase oligomerization in muscle tissue. Kidneys heavily depend on the oxidative phosphorylation in mitochondria. Here we report kidney-related phenotypes in aged Mtln knockout mice. Similar to Mtln knockout mice muscle mitochondria, those of the kidney demonstrate a decreased respiratory complex I activity and excessive cardiolipin damage. Aged male mice carrying Mtln knockout demonstrated an increased frequency of renal proximal tubules' degeneration. At the same time, a decreased glomerular filtration rate has been more frequently detected in aged female mice devoid of Mtln. An amount of Mtln partner protein, Cyb5r3, is drastically decreased in the kidneys of Mtln knockout mice.

Mitoregulin Contributes to Creatine Shuttling and Cardiolipin Protection in Mice Muscle.

Small peptides compose a large share of the mitochondrial proteome. Mitoregulin (Mtln) is a mitochondrial peptide known to contribute to the respiratory complex I functioning and other processes in mitochondria. In our previous studies, we demonstrated that Mtln knockout mice develop obesity and accumulate triglycerides and other oxidation substrates in serum, concomitant with an exhaustion of tricarboxylic acids cycle intermediates. Here we examined the functional role of Mtln in skeletal muscles, one of the major energy consuming tissues. We observed reduced muscle strength for Mtln knockout mice. Decrease of the mitochondrial cardiolipin and concomitant increase in monolysocardiolipin concentration upon Mtln inactivation is likely to be a consequence of imbalance between oxidative damage and remodeling of cardiolipin. It is accompanied by the mitochondrial creatine kinase octamer dissociation and suboptimal respiratory chain performance in Mtln knockout mice.

A Novel C1q Domain-Containing Protein Isolated from the Mollusk Modiolus kurilensis Recognizing Glycans Enriched with Acidic Galactans and Mannans.

C1q domain-containing (C1qDC) proteins are a group of biopolymers involved in immune response as pattern recognition receptors (PRRs) in a lectin-like manner. A new protein MkC1qDC from the hemolymph plasma of Modiolus kurilensis bivalve mollusk widespread in the Northwest Pacific was purified. The isolation procedure included ammonium sulfate precipitation followed by affinity chromatography on pectin-Sepharose. The full-length MkC1qDC sequence was assembled using de novo mass-spectrometry peptide sequencing complemented with N-terminal Edman's degradation, and included 176 amino acid residues with molecular mass of 19 kDa displaying high homology to bivalve C1qDC proteins. MkC1qDC demonstrated antibacterial properties against Gram-negative and Gram-positive strains. MkC1qDC binds to a number of saccharides in Ca2+-dependent manner which characterized by structural meta-similarity in acidic group enrichment of galactose and mannose derivatives incorporated in diversified molecular species of glycans. Alginate, κ-carrageenan, fucoidan, and pectin were found to be highly effective inhibitors of MkC1qDC activity. Yeast mannan, lipopolysaccharide (LPS), peptidoglycan (PGN) and mucin showed an inhibitory effect at concentrations three orders of magnitude greater than for the most effective saccharides. MkC1qDC localized to the mussel hemal system and interstitial compartment. Intriguingly, MkC1qDC was found to suppress proliferation of human adenocarcinoma HeLa cells in a dose-dependent manner, indicating to the biomedical potential of MkC1qDC protein.

Immune state correlates with histopathological level and reveals molluscan health in populations of Modiolus kurilensis by integral health index (IHI).

Quantitative analysis of the histopathological and immune parameters of bivalve Modiolus kurilensis collected from water areas with different level of ecotoxicological stress was performed. Significant differences between samples from polluted and non-polluted sites were revealed for total haemocyte count; percentage of agranulocytes; size and internal complexity of agranulocytes and granulocytes; phagocytic activity; percentage of NBT-positive cells; hemolytic activity and plasma protein concentration; percentage of the optical density of haemolymph major polypeptide bands at 55 kDa, 78 kDa, and 124 kDa; concretion coverage area in the kidney tubules; thickness of the tubular basement membrane; nephrocyte shape; and karyopyknosis of the kidneys; and hypervacuolisation; necrosis; karyopyknosis; haemocyte infiltration; fibrosis; and invasion of the digestive gland. Analysis of the global histopathological condition index based on the weighted indices also revealed that both the digestive gland and kidneys showed significantly greater histopathological changes in the bivalves collected from polluted water. Bivalve histopathology is an established tool in aquatic toxicology. However, it reflects a morphological picture of change, which, as a rule, can be clearly recorded only at the later stages of pathology, and in some cases, indicates an adaptation to stressors within the physiological norm. In this respect, a promising and highly sensitive biomarker of the functional state of bivalves, in terms of norm and pathology as well as their habitat, is the evaluation of immune status in combination with morphological changes. However, the use of different methods and scales of assessment and the diagnosis of biomarkers, characterised by different profiles of the stress response, makes it difficult to compare the results of different studies. We propose a reliable and powerful system for assessing the physiological state of bivalve molluscs, expressed in the integral health index (IHI) and based on the standardisation of the numerical values for all parameters that have significant differences between animals collected from impacted and non-impacted water areas. In our study, IHI calculated in three variants (for histopathological parameters, for immunological parameters, and in combination) showed the most significant differences in each of the cases, but the strongest difference (-4.07) was in calculating the total IHI, which included both the immune and histopathological parameters (p = 0.00005).